Nontypeable Haemophilus influenzae (NTHi) commonly colonize the mucosal surfaces of the human upper respiratory tract. For the most part, this species resides in that environment in a benign (symbiotic?) relationship with the human host. With pathophysiologic changes (obstruction of outflow ducts, loss of the mucociliary elevator, viral infection, etc.) in the airway anatomy and mucosal surface, NTHi can cause human upper and lower tract respiratory infections. The serum resistant phenotype appears to be a prerequisite for NTHi to initiate systemic infection (acute bronchitis, otitis media, etc). Studies from our laboratory and others have shown that sialic acid is important in the biology of many NTHi strains. Sialylation of LOS increases resistance to the bactericidal effect of normal human serum and is an important constituent of the NTHi biofilm. NTHi cannot synthesize sialic acid and must acquire it from its environment. Free sialic acid is quite limited in the human respiratory tract while sialoglycoproteins, such as mucin, are abundant. In our progress report, we have demonstrated that NTHi can remove sialic acid from mucin and incorporate this sugar into its LOS. The mechanism by which this is accomplished is unknown. Analysis of the 13 annotated NTHi genomes indicate that there are no genes homologous to any known eukaryotic or prokaryotic neuraminidase. We have also found that about 40% of NTHi strains do not incorporate sialic acid into their LOS. The factors determining NTHi LOS-based serum resistance and sensitivity are poorly defined. Better definition may lead to novel vaccines approaches. We have recently performed an NTHi human colonization experiment with NTHi 2019strR. The NTHi 2019strR strains recovered from 4 patients six days after infection demonstrated genomic changes after 454 pyrosequencing of these strains and NTHI 2019strR. The hypotheses underlying the studies in this proposal are: A) NTHi can scavenge sialic acid from human mucin by a unique glycosidase system, B) the LOS is a major determinant of serum resistance in NTHi including strains which do not sialylate their LOS, C) NTHi residence in the human nasopharynx selects for specific LOS phase variants and D) the transformability of NTHi allows introduction of foreign DNA into the NTHi genome. The following specific aims are proposed to resolve these hypotheses: 1) Identification of the mechanism by which sialic acid is removed from human mucin, 2) Comparative analysis of the LOS of selected serum resistant NTHi and serum sensitive NTHi strains which cannot sialylate their LOS to determine if specific LOS glycoforms are the basis of serum sensitivity and 3) Analysis of the phase-variation state of genes involved in LPS biosynthesis and the uptake and expression of foreign DNA in NTHi strain 2019strR recovered from patients during human colonization experiments.